Bridging Project 1: Conformational Transitions in P-class ATPases

P类（或E1-E2型）ATP酶构成阳离子转运酶的超家族，存在于原核生物和真核生物中，其成员介导所有常见生物相关阳离子的膜通量[1]。P类泵使用ATP逆梯度输送离子。肌浆网Ca+ -ATP酶（SERCA）通过交换H”"将2Ca+从肌细胞的胞浆泵到肌浆网。在每个正常的循环中，Na/K泵将3 Na”运出细胞，通过2 K“运入细胞。 以一个ATP分子的水解为代价。这个关于P类ATP酶的扩展桥梁项目代表了两个桥梁项目的组合，重点是Na/K和SERCA泵。 根据SERCA的晶体学快照和最近解决的Na/K泵的第一个晶体结构，我们现在处理一系列显着的图片，显示这些酶如何看待它们的运输循环的不同状态。目前，SERCA是迄今为止功能上最不同的构象已被描述在精确的结构细节的膜蛋白[4，5]。然而，SERCA的电生理学研究比Na/K泵更难进行，Na/K泵可以在细胞表面异源表达。由于这个原因，虽然关于Na/K泵的结构信息更加有限（只有一个X射线结构可用），但最有信息的功能分析来自Na/K泵的实验。 在这个建议中，我们解决的Na/K和SERCA泵的问题：什么是参与的构象动力学的泵转换通过构象状态揭示的X射线晶体学？ ATP的结合、磷酸化和带电物质在蛋白质核心的运动之间的耦合性质是什么？什么是逐步电压敏感步骤？在不同的生理条件下，完整的反应途径是什么？对这些问题的回答将使结构变化与膜P类ATP酶泵的功能相关联。

P-class (or E1-E2-type) ATPases constitute a superfamily of cation transport enzymes, present both in prokaryote and eukaryote, whose members mediate membrane flux of all common biologically relevant cations [1]. P-class pumps use ATP to transport ions against a gradient. The sarcoplasmic reticulum Ca+ -ATPase (SERCA) pumps 2 Ca+ from the cytosol of muscle cells to the sarcoplasmic reticulum by exchanging H"". In each normal cycle, the Na/K pump transports 3 Na" out of the cell by 2 K = into the cell at the expense of the hydrolysis of one molecule of ATP. This extended bridge project about P-class ATPases represents the combination of two bridge projects, focused on the Na/K and SERCA pumps. From crystallographic snapshots of SERCA and the recently solved first crystal structure of the Na/K pump, we now dispose of a remarkable series of pictures showing how these enzymes look at different states of their transport cycle. SERCA is now by far the membrane protein where the most functionally different conformations have been described in precise structural detail [4, 5]. However, electrophysiological studies with SERCA are harder to do than with the Na/K pump, which can be expressed heterologously on the surface of cells. For this reason, while structural information about the Na/K pump is more limited (with only one x-ray structure available), the most informative functional analysis comes from experiments on the Na/K pump. In this proposal, we address questions about the Na/K and SERCA pumps: What are the conformational dynamics involved as the pump transits through conformational states revealed by x-ray crystallography? What is the nature of the coupling between the binding of ATP, phosphorylation, and the movements of charged species across the core of the protein? What are the stepwise voltage-sensitive steps? What is the complete reaction pathway and what are the individual transition rates under various physiological conditions? Answers to these questions will correlate the structural changes to the function of membrane P-class ATPase pumps.